Role of Cathepsin S in Periodontal Wound Healing–An in Vitro Study on Human PDL Cells Svenja Memmert1,2* , Marjan Nokhbehsaim1, Anna Damanaki1, Andressa V

Memmert et al. BMC Oral Health (2018) 18:60 https://doi.org/10.1186/s12903-018-0518-2

RESEARCH ARTICLE Open Access Role of cathepsin S In periodontal wound healing–an in vitro study on human PDL cells Svenja Memmert1,2* , Marjan Nokhbehsaim1, Anna Damanaki1, Andressa V. B. Nogueira3, Alexandra K. Papadopoulou4, Christina Piperi5, Efthimia K. Basdra5, Birgit Rath-Deschner2, Werner Götz2, Joni A. Cirelli3, Andreas Jäger2 and James Deschner1,6

Abstract Background: Cathepsin S is a cysteine protease, which is expressed in human periodontal ligament (PDL) cells under inflammatory and infectious conditions. This in vitro study was established to investigate the effect of cathepsin S on PDL cell wound closure. Methods: An in vitro wound healing assay was used to monitor wound closure in wounded PDL cell monolayers for 72 h in the presence and absence of cathepsin S. In addition, the effects of cathepsin S on specific markers for apoptosis and proliferation were studied at transcriptional level. Changes in the proliferation rate due to cathepsin S stimulation were analyzed by an XTT assay, and the actions of cathepsin S on cell migration were investigated via live cell tracking. Additionally, PDL cell monolayers were treated with a toll-like receptor 2 agonist in the presence and absence of a cathepsin inhibitor to examine if periodontal bacteria can alter wound closure via cathepsins. Results: Cathepsin S enhanced significantly the in vitro wound healing rate by inducing proliferation and by increasing the speed of cell migration, but had no effect on apoptosis. Moreover, the toll-like receptor 2 agonist enhanced significantly the wound closure and this stimulatory effect was dependent on cathepsins. Conclusions: Our findings provide original evidence that cathepsin S stimulates PDL cell proliferation and migration and, thereby, wound closure, suggesting that this cysteine protease might play a critical role in periodontal remodeling and healing. In addition, cathepsins might be exploited by periodontal bacteria to regulate critical PDL cell functions. Keywords: cathepsin S, periodontal ligament cells, wound closure, migration

Background Fusobacterium nucleatum induce an inflammatory host Periodontitis is a highly-prevalent chronic inflammatory response through binding to special receptors such as disease characterized by bone, attachment and even toll-like receptor (TLR) 2, which can ultimately result in tooth loss [1]. Oral periodontopathogens are a pre- the destruction of periodontal structures [3]. requisite for the disease but not sufficient to induce peri- Periodontal ligament (PDL) cells are resident cells of odontitis. Additional risk factors, such as smoking, the periodontium and have a critical role in tissue genetic predisposition and certain systemic diseases con- homeostasis, destruction and regeneration by their abil- tribute to the initiation and progression of periodontitis ity to synthesize and degrade collagen and other matrix [2]. The periodontal microorganisms such as molecules [4]. However, these cells can also participate in the immunoinflammatory processes of periodontitis * Correspondence: [email protected] [5]. Periodontal healing is determined by the type of cells 1Section of Experimental Dento-Maxillo-Facial Medicine, Center of Dento-Maxillo-Facial Medicine, University of Bonn, Bonn, Germany that repopulate the root. By the application of regenera- 2Department of Orthodontics, Center of Dento-Maxillo-Facial Medicine, tive treatment methods, which promote PDL cell prolif- University of Bonn, Bonn, Germany eration, migration and attachment, the re-establishment Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Memmert et al. BMC Oral Health (2018) 18:60 Page 2 of 7

of the initial periodontal tissue architecture is possible used for experiments at 60%-100% confluence, depend- [6]. However, the outcomes of currently available regen- ing on the individual protocol of each experiment [22]. erative treatment approaches are sometimes compromised by a number of factors and are not predictable [7, 8]. Cell treatment Therefore, the search for new molecules with a regenera- One day prior to the experiments, the FBS concentration tive potential are a major goal in periodontology [9]. was reduced to 1%. PDL cells were incubated with the Cathepsin S (CTSS) is a lysosomal cysteine protease active form of CTSS (1 ng/μl; activity 143.4 U/mL; and has the ability to remain stable and active under Calbiochem, San Diego, CA, USA) for up to 72 h [24]. neutral pH [10–12]. Therefore, it can evoke both intra- In a subset of experiments, PDL cells were incubated and extracellular activities. Intracellularly, CTSS func- with a TLR2 agonist (1 μg/ml; Pam3CSK4; Invivogen, tions as a processing enzyme and is critical for protein San Diego, CA, USA) in the presence and absence of a trafficking and secretion, while extracellularly it has a cathepsin inhibitor (50 μM; Z-FA-FMK; Santa Cruz pivotal role in tissue remodeling [11]. This protease has Biotechnology, Dallas, TX, USA) for 24 h [25, 26]. the capacity to degrade multiple components of the extracellular matrix, such as collagen, elastin, fibronec- In vitro wound healing assay tin, laminin and proteolglycans [11, 13, 14]. Moreover, To analyze the in vitro wound healing, a well-established substrates of CTSS not only comprise antigenic as well in vitro wound healing model was applied as in our pre- as antimicrobial peptides but also play a fundamental vious studies [27–29]. Briefly, PDL cells were cultured role in antigen processing and presentation [11, 15, 16]. on 35 mm plastic culture dishes and grown to 100% Additionally, it has been shown that CTSS promotes cell confluence. One day after reduction of FBS concentra- migration [17]. Hence, these functions of CTSS suggest tion, a 3–4 mm-wide wound was inflicted in a standard- a complex role in immunoinflammatory diseases and ized manner so that cell free areas were created in the healing processes [14, 18, 19]. cell monolayers. Afterwards, all non-adherent cells were CTSS is not produced ubiquitously and its synthesis removed through multiple washing steps with DMEM. seemed to be restricted to immunocompetent cells, The wounded monolayers were then cultured in the such as macrophages, lymphocytes and dendritic cells presence and absence of CTSS for 72 h or the TLR2 [14, 19]. Previously, we have found that CTSS is also agonist in combination with or without the cathepsin secreted by PDL cells and that its synthesis is regulated by inhibitor for 24 h, as described above. By using a JuLI™ inflammatory and microbial stimuli, suggesting strongly a Br and the JuLI™ Br PC software (both NanoEnTek, Seoul, role of this protease in oral inflammatory diseases [20]. Korea), the wound closure was monitored over time and, Moreover, in gingival biopsies from sites of periodontitis, subsequently, the wound healing rates were calculated. CTSS was identified as a hub protein in the protein- protein interaction network of differentially expressed Cell migration genes, also suggesting an involvement of CTSS in periodon- Live cell imaging was applied to monitor cell migration titis [21]. Therefore, the aim of this in vitro study was to in- over a period of 24 h. Cells were grown to 100% conflu- vestigate the effects of CTSS on PDL cell wound closure. ence, and cell free areas were created in a standardized manner. Subsequently, cells were treated as described above. The live cell tracker JuLI™ Br device and the JuLI™ Methods Br PC software were used to capture images and to Isolation and characterization of PDL cells monitor the migration of individual cells. In each group Written informed consent and approval of the Ethics and donor, 6 cells which had moved the farthest into the Committee of the University of Bonn were obtained cell-free area after 24 h, were marked and traced. Images (#117/15). Human PDL cells were taken from caries-free were transferred to and analyzed by the freely available and periodontally healthy teeth of 5 donors (mean age: image-processing software Image J 1.43 [30]. 14.6 years, min/max: 13/19 years; 3 males/2 females), who had to undergo tooth extractions for orthodontic Gene expression reasons [22, 23]. Cells were harvested from the medial PDL cell monolayers were cultured in the presence and part of the tooth root and grown in Dulbecco’s minimal absence of CTSS for 24 h. Afterwards, RNA extraction essential medium (DMEM, Invitrogen, Karlsruhe, was performed with a commercially available RNA Germany) supplemented with 10% fetal bovine serum extraction kit (RNeasy Protect Minikit, Qiagen, Hilden, (FBS, Invitrogen), 100 units penicillin and 100 μg/mL Germany) and 1 μg of RNA was converted into cDNA streptomycin (Invitrogen) in a humidified atmosphere of by reverse-transcription with the IScript Select cDNA 5% CO2 at 37 °C. Cells between passages 3 to 5 were Synthesis Kit (Bio-Rad Laboratories, Munich, Germany). phenotyped according to Basdra and Komposch and Expressions of Proliferating Cell Nuclear Antigen Memmert et al. BMC Oral Health (2018) 18:60 Page 3 of 7

(PCNA) and p53, which are markers for cell prolifera- When the average wound closure over 72 h was cal- tion and cell death, respectively, were determined by culated, the wound fill rate in the CTSS-treated real-time PCR by using the iCyler iQ detection system group and control were 49% and 29%, respectively, (Bio-Rad Laboratories) [31, 32]. cDNA expression of demonstrating a significant difference between both 1 μl of cDNA was detected via real-time PCR in a 25 μl groups (Fig. 1c). reaction mixture containing 2.5 μl respective QuantiTect Primer assay (Qiagen), 12.5 μl QuantiTect SYBR Green Influence of CTSS on migration Master Mix (Qiagen) and 9 μl of nuclease free water, as The wound fill rate is also determined by the speed of recommended by the manufacturer. The protocol com- cell migration. Therefore, we also studied the effects of prised a heating phase at 95 °C for 5 min to activate the CTSS on this cell function. In wounded PDL cell mono- enzyme, followed by 40 cycles of a denaturation step at layers in the presence and absence of CTSS, the cell mi- 95 °C for 10 s and a combined annealing/extension step gration was monitored by live cell imaging over a period at 60 °C for 30 s. Melting point analysis was performed of 24 h. Cells which had moved the farthest into the after each run. For normalization, the housekeeping gene cell-free area after 24 h, were marked and traced. As GAPDH was used. shown in Fig. 2a, CTSS treatment of cells resulted in an accelerated migration, as compared to control. For the Cell proliferation majority of time points, the distance which the cells cov- Cell proliferation was analyzed by using the AppliChem ered within 1 h, was greater in CTSS-treated groups Cell Proliferation Kit XTT (AppliChem, Darmstadt, than in control. When the average speed over 24 h was Germany). In a 96-well-plate, PDL cells (5.000/well) calculated, the migration was significantly faster in the μ μ were grown to 60% confluence and stimulated with CTSS group (27.16 m/h) than in control (22.34 m/h), CTSS for up to 24 h. XTT reaction solution was added as depicted in Fig. 2b. to the medium 4 h before measurements. Finally, the absorbance was determined with a microplate reader Actions of CTSS on proliferation and apoptosis (PowerWave x, BioTek Instruments, Winooski, VT, Since wound closure also depends on the cell number, USA) at 475 nm. which is determined by proliferation and apoptosis, we also studied the CTSS actions on these cell functions. Treatment of PDL cells with CTSS resulted in a signifi- Statistical Analysis cantly increased expression of PCNA, a specific marker The IBM SPSS Statistics software (Version 22, IBM of cell proliferation (Fig. 3a). However, no difference be- SPSS, Chicago, IL, USA) was used for statistical ana- tween groups was observed with regard to p53 expres- lysis. Mean values and standard errors of the mean sion, a marker of apoptosis (Fig. 3a). To confirm our (SEM) were calculated for quantitative data. All ex- observation for PCNA at transcriptional level, we also periments were performed in triplicate and repeated studied the proliferation of PDL cells in the presence at least twice. For statistical comparison of the and absence of CTSS by using a commercially available groups, the t- and Mann-Whitney-U tests were ap- XTT assay. Although there was a trend to an increased plied. Differences between groups were considered proliferation in the CTSS group, the difference did not p significant at < 0.05. reach significance at 4 h (Fig. 3b). However, incubation of PDL cells with CTSS for 24 h induced a significantly Results enhanced proliferation rate by 27% (Fig. 3b). Effect of CTSS on wound healing SincewerecentlyfoundthatPDLcellsproduceCTSS Involvement of cathepsins in TLR2 effects on wound and other studies have demonstrated that CTSS af- closure fects cell proliferation and migration, we analyzed The aforementioned experiments demonstrated that CTSS effects on wound closure of PDL cell mono- CTSS accelerates wound closure, and our previous ex- layers [17, 18, 20]. After incubation of wounded PDL periments had revealed that F. nucleatum, which is able cell monolayers with CTSS, the wound closure was to activate TLR2, upregulates CTSS [20]. Therefore, we followed up over 72 h. Wounded monolayers without next sought to examine whether a TLR2 agonist would CTSS treatment served as control. As shown in Fig. enhance the wound closure and whether such a poten- 1a and b, wound closure started earlier in the CTSS- tial stimulatory effect would involve cathepsins. As treated group as compared to control. Moreover, the shown in fig. 4a, incubation of PDL cell monolayers with wound fill rate was higher in the CTSS-treatment a TLR2 agonist enhanced significantly the average groupatalltimepoints.At72h,thewoundclosure wound closure by approximately 70% over 24 h. How- was 79% in the treatment group and 58% in control. ever, when the TLR2 agonist-treated monolayers were Memmert et al. BMC Oral Health (2018) 18:60 Page 4 of 7

Fig. 1 a Wound closure of PDL cell monolayers in the presence or absence of CTSS (1 ng/μl) over 72 h. The wound closure, i.e., the percentage of cell coverage of the initially cell-free zones created by wounding, were analyzed by live cell imaging. Mean ± SEM. b Wound closure of PDL cell monolayers in the presence or absence of CTSS (1 ng/μl) at 0 h, 24 h, 48 h and 72 h. Images from one representative donor are shown. c Average wound closure of PDL cell monolayers shown in a. Mean ± SEM (n = 26), * significant (p < 0.05) difference between groups simultaneously exposed to a cathepsin inhibitor, the en- lysosomal cysteine protease capable of degrading com- hanced wound closure was significantly reduced by 50% ponents of the extracellular matrix, such as collagen, (Fig. 4b). elastin, fibronectin, laminin and proteoglycans, suggesting a pivotal role in tissue homeostasis and repair Discussion [11, 13, 14]. This assumption is further supported by The present study investigated the effects of CTSS on the observation that CTSS promotes cell migration PDL cell wound closure. Our findings provide original and, additionally, regulates osteoblast differentiation evidence that CTSS stimulates PDL cell proliferation and bone remodeling [17, 18, 34]. The synthesis of and migration and, thereby, wound closure, suggesting CTSS by periodontal cells and its role in periodontal that this cysteine protease might play a critical role in tissues has been neglected so far. Our previous exper- periodontal remodeling and healing. Moreover, TLR2 iments have provided novel evidence that PDL cells activation also accelerates the PDL cell wound closure can produce this cysteine protease [20]. Periodontal and this stimulatory effect depends on cathepsins, sug- therapy results in wounding of the periodontal tissues gesting that cathepsins might be exploited by periodon- and, subsequently, wound healing is initiated. Ideally, tal bacteria to regulate critical PDL cell functions. the healing is characterized by the restoration of the CTSS has been broadly implicated in health and path- original tissue structure, form and function. However, ology including autoimmune diseases, allergic inflamma- periodontal regeneration requires appropriate PDL tion and asthma, diabetes and obesity, cardiovascular cell proliferation and migration. Therefore, in the and pulmonary diseases as well as cancer [33]. CTSS is a present study, we sought to investigate possible Memmert et al. BMC Oral Health (2018) 18:60 Page 5 of 7

Fig. 2 a PDL cell migration, i.e., the distance which the cells covered μ within 1 h, in the presence or absence of CTSS (1 ng/ l) over 24 h. Fig. 4 a Average wound closure of PDL cell monolayers in the n Mean ± SEM ( = 12). b Average speed of PDL cells shown in a. presence or absence of a TLR2 agonist (Pam3CSK4; 1 μg/ml) over p Mean ± SEM, * significant ( < 0.05) difference between groups 24 h. Mean ± SEM. * significant (p < 0.05) difference between groups. b Average wound closure of PDL cell monolayers treated with a TLR2 agonist in the presence and absence of a cathepsin inhibitor (Z-FA-FMK; 50 μM) over 24 h. Mean ± SEM, * significant (p < 0.05) difference between groups

actions of CTSS on PDL cells with a special focus on proliferation, migration and wound healing in vitro. The present experiments revealed that these cell functions are regulated by CTSS. The cysteine protease caused a significant upregulation of PCNA, a specific marker of proliferation, and proliferation, as assessed by an XTT assay. Moreover, our experiments showed that CTSS also increased the speed of PDL cell migration. Since both proliferation and migration deter- mine the would fill rate, our observation that CTSS promotes wound healing is supported by these findings. Gonzales et al. studied the gene expression in gingival biopsies from Rhesus monkeys and advocated a role for CTSS in periodontitis [35]. Furthermore, CTSS was identified as a hub protein in the protein-protein interaction network of differentially expressed genes, also indicating an involvement of this protease in periodontal diseases [21]. The results of our previous study which showed an increased production of CTSS by periodontal Fig. 3 a PCNA and p53 gene expressions in the presence or cells in response to inflammatory and infectious stimuli absence of CTSS (1 ng/μl) at 24 h. Mean ± SEM (n = 9), * significant concur very well with these reports [20]. However, the (p < 0.05) difference between groups. b PDL cell proliferation in the findings of the present study demonstrate that CTSS μ presence or absence of CTSS (1 ng/ l) at 4 h and 24 h. Mean ± SEM might not only play a role in periodontal tissue destruc- (n = 24), * significant (p < 0.05) difference between groups tion but also healing. Memmert et al. BMC Oral Health (2018) 18:60 Page 6 of 7

Notably, exposure of PDL cell monolayers to the TLR2 phenotype. Future studies should also examine the agonist resulted in a significantly accelerated wound actions of CTSS on other periodontal cells involved in closure in our study. Further experiments revealed that periodontal homeostasis and repair. Moreover, in order the stimulation of the in vitro wound healing by the to further explore the role of CTSS in a more complex TLR2 agonist was dependent on cathepsins. Interest- environment, an experimental periodontitis model in ingly, inhibition of TLR2 has been shown to reduce the CTSS knock-out mice might be helpful. CTSS gene expression in human endothelial cells, sup- porting our data [36]. Our observation is of major im- Conclusions portance, because it links the experiments of this study In summary, our study investigated the actions of CTSS with our previous findings [20]. Moreover, since we used on PDL cell wound closure. Our findings provide a TLR2 agonist and an unspecific cathepsin inhibitor, original evidence that cathepsin S stimulates PDL cell this result may have an even broader significance for the proliferation and migration and, thereby, wound closure, understanding of periodontal diseases, because peri- suggesting that this cysteine protease might play a crit- odontitis is not only caused by a single bacterium or ical role in periodontal remodeling and healing. In mediated by a single member of the cathepsin family. addition, cathepsins might be exploited by periodontal Whether the increased wound closure in the presence of bacteria to regulate critical PDL cell functions. the TLR2 agonist, as observed in our experiments, might Abbreviations be an attempt of the cells to maintain tissue homeosta- CTSK: Cathepsin K; CTSS: Cathepsin S; DMEM: Dulbecco’s minimal essential sis, has to be clarified in further studies. medium; FBS: Fetal bovine serum; PCNA: Proliferating cell nuclear antigen; Cathepsin K (CTSK), another member of the cathepsin PDL: Periodontal ligament; SEM: Standard errors of the mean; TLR: Toll-like receptor. family, has also been associated with periodontal diseases [37–39]. Interestingly, CTSS has the ability to Acknowledgements degrade CTSK, indicating complex interactions between The authors would like to thank Ms. Ramona Menden, Ms. Silke van Dyck, Ms. Inka Bay and Prof. Heiko Spallek for their valuable support. both cathepsins [40]. Further research should also focus on the role of CTSK in PDL cell proliferation, migration Funding and wound closure. This study was supported by the Medical Faculty of the University of Bonn, the University of Sydney, the German Orthodontic Society (DGKFO) and the Although not investigated in our study, the stimulatory German Research Foundation (DFG, ME 4798/1–1). effects of CTSS on cell migration and proliferation might involve TLR2-mediated p38/Akt signaling activa- Availability of data and materials The datasets used and/or analyzed during the current study are available tion and histone deacetylase 6, as it has been disclosed from the corresponding author on reasonable request. in vascular smooth muscle cells by Wu and colleagues [18]. Another study on macrophages suggests that CTSS Authors’ contributions SM, CP, EB, WG, JC, AJ and JD made substantial contributions to conception promotes cell migration through degradation of elastic and design. SM, MN, AN and JD substantially contributed to the acquisition fiber integrity [17]. Furthermore, CTSS might influence of data. SM, AD, AP, BRD and JD substantially contributed to interpretation of proliferation of PDL cells via peroxisome proliferator- data and analysis. SM, MN, AD, AN, AP, CP, EB, BRD, WG, JC, AJ and JD have been involved in drafting the manuscript or revising it critically for important activated receptor-gamma, as revealed in human umbil- intellectual content. SM, MN, AD, AN, AP, CP, EB, BRD, WG, JC, AJ and JD ical vein endothelial cells [41]. Further studies should have given final approval of the version to be published. SM, MN, AD, AN, focus on the mechanisms underlying the stimulatory AP, CP, EB, BRD, WG, JC, AJ and JD agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of effects of CTSS on wound healing in periodontal cells. any part of the work are appropriately investigated and resolved. All authors In the present study, CTSS was used at concentrations read and approved the final manuscript. of 1 ng/μl, as used in our previous experiments and in Ethics approval and consent to participate studies by other investigators [24]. Furthermore, we Approval of the Ethics Committee of the University of Bonn was obtained (#117/15). applied an established vitro wound healing model which All donors of the PDL cells or their parents gave written informed consent. is commonly used [27–29, 42, 43]. Nonetheless, the limi- Consent for publication tation of any in vitro model, including this one, is that it Not applicable. cannot fully mimic the plethora of actions and interactions of different cell and tissue types that take place in Competing interests The authors declare that they have no competing interest. vivo [44]. Our experiments focussed on PDL cells, which constitute a heterogenous cell population, because they Publisher’sNote are critical for both periodontal destruction and Springer Nature remains neutral with regard to jurisdictional claims in regeneration. By phenotyping the cells before our experi- published maps and institutional affiliations. ments, we confirmed their ability to differentiate into Author details osteoblastic cells. But as no osteogenic medium was ap- 1Section of Experimental Dento-Maxillo-Facial Medicine, Center of plied in the experiments, the cells acquired a fibroblastic Dento-Maxillo-Facial Medicine, University of Bonn, Bonn, Germany. Memmert et al. BMC Oral Health (2018) 18:60 Page 7 of 7

2Department of Orthodontics, Center of Dento-Maxillo-Facial Medicine, JA, Jäger A, Deschner J. Role of Cathepsin S in Periodontal Inflammation University of Bonn, Bonn, Germany. 3Department of Diagnosis and Surgery, and Infection. Mediat Inflamm. 2017;2017:4786170. School of Dentistry at Araraquara, Sao Paulo State University, UNESP, 21. Song L, Yao J, He Z, Xu B. Genes related to inflammation and bone loss Araraquara, Brazil. 4Discipline of Orthodontics, Faculty of Dentistry, University process in periodontitis suggested by bioinformatics methods. BMC Oral of Sydney, Sydney, Australia. 5Department of Biological Chemistry, Medical Health. 2015;15:105. School, National and Kapodistrian University of Athens, Athens, Greece. 6Noel 22. Basdra EK, Komposch G. Osteoblast-like properties of human periodontal Martin Visiting Chair, Faculty of Dentistry, University of Sydney, Sydney, ligament cells: an in vitro analysis. Eur J Orthod. 1997;19:615–21. Australia. 23. Mariotti A, Cochran DL. Characterization of fibroblasts derived from human periodontal ligament and gingiva. J Periodontol. 1990;61:103–11. Received: 25 October 2017 Accepted: 20 March 2018 24. Zou F, Schäfer N, Palesch D, Brücken R, Beck A, Sienczyk M, Kalbacher H, Sun Z, Boehm BO, Burster T. Regulation of cathepsin G reduces the activation of proinsulin-reactive T cells from type 1 diabetes patients. PLoS One. 2011;6:e22815. References 25. Andrukhov O, Hong JS, Andrukhova O, Blufstein A, Moritz A, Rausch-Fan X. – 1. Slots J. Periodontitis: facts, fallacies and the future. Periodontol. 2017;75:7 23. Response of human periodontal ligament stem cells to IFN-γ and TLR- 2. Tatakis DN, Kumar PS. Etiology and pathogenesis of periodontal diseases. agonists. Sci Rep. 2017;7:12856. – Dent Clin North Amer. 2005;49:491 516. 26. Schotte P, Schauvliege R, Janssens S, Beyaert R. The cathepsin B inhibitor z- 3. Nogueira AV, Nokhbehsaim M, Eick S, Bourauel C, Jäger A, Jepsen S, Cirelli FA.fmk inhibits cytokine production in macrophages stimulated by JA, Deschner J. Regulation of visfatin by microbial and biomechanical lipopolysaccharide. J Biol Chem. 2001;276:21153–7. – signals in PDL cells. Clin Oral Investig. 2014;18:171 8. 27. Nokhbehsaim M, Winter J, Rath B, Jäger A, Jepsen S, Deschner J. Effects of 4. Kaneda T, Miyauchi M, Takekoshi T, Kitagawa S, Kitagawa M, Shiba H, enamel matrix derivative on periodontal wound healing in an inflammatory Kurihara H, Takata T. Characteristics of periodontal ligament subpopulations environment in vitro. J Clin Periodontol. 2011;38:479–90. obtained by sequential enzymatic digestion of rat molar periodontal 28. Nokhbehsaim M, Deschner B, Bourauel C, Reimann S, Winter J, Rath B, Jäger – ligament. Bone. 2006;38:420 6. A, Jepsen S, Deschner J. Interactions of enamel matrix derivative and 5. Konermann A, Stabenow D, Knolle PA, Held SA, Deschner J, Jäger A. biomechanical loading in periodontal regenerative healing. J Periodontol. Regulatory role of periodontal ligament fibroblasts for innate immune cell 2011;82:1725–34. – function and differentiation. Innat Immun. 2012;18:745 52. 29. NokhbehsaimM,KeserS,JägerA,JepsenS,DeschnerJ.Regulationof 6. Lin NH, Menicanin D, Mrozik K, Gronthos S, Bartold PM. Putative stem cells regenerative periodontal healing by NAMPT. Mediat Inflamm. 2013;2013:202530. – in regenerating human periodontium. J Periodontal Res. 2008;43:514 23. 30. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of 7. Deschner J, Nokhbehsaim M. Regulatory effects of inflammatory and image analysis. Nat Methods. 2012;9:671–5. biomechanical signals on regenerative periodontal healing. Int J Oral 31. Li K, Wu D, Chen X, Zhang T, Zhang L, Yi Y, Miao Z, Jin N, Bi X, Wang H, Xu – Maxillofac Implants. 2013;28:e472 7. J, Wang D. Current and emerging biomarkers of cell death in human 8. Miron RJ, Sculean A, Cochran DL, Froum S, Zucchelli G, Nemcovsky C, disease. Biomed Res Int. 2014;2014:690103. Donos N, Lyngstadaas SP, Deschner J, Dard M, Stavropoulos A, Zhang Y, 32. Dietrich DR. Toxicological and pathological applications of proliferating cell Trombelli L, Kasaj A, Shirakata Y, Cortellini P, Tonetti M, Rasperini G, Jepsen nuclear antigen (PCNA), a novel endogenous marker for cell proliferation. S, Bosshardt DD. Twenty years of enamel matrix derivative: the past, the Crit Rev Toxicol. 1993;23:77–109. – present and the future. J Clin Periodontol. 2016;43:668 83. 33. Wilkinson RD, Williams R, Scott CJ, Burden RE. Cathepsin S: therapeutic, 9. Hynes K, Menicanin D, Gronthos S, Bartold PM. Clinical utility of stem cells diagnostic, and prognostic potential. Biol Chem. 2015;396:867–82. – for periodontal regeneration. Periodontol. 2012;59:203 27. 34. Rauner M, Föger-Samwald U, Kurz MF, Brünner-Kubath C, Schamall D, 10. Chapman HA Jr, Munger JS, Shi GP. The role of thiol proteases in tissue Kapfenberger A, Varga P, Kudlacek S, Wutzl A, Höger H, Zysset PK, Shi GP, – injury and remodeling. Am J Respir Crit Care Med. 1994;150:155 9. Hofbauer LC, Sipos W, Pietschmann P. Cathepsin S controls adipocytic and 11. Dickinson DP. Cysteine paptodases of mammals: their biological roles and osteoblastic differentiation, bone turnover, and bone microarchitecture. potential effects in the oral cavity and other tissues in health and disease. Bone. 2014;64:281–7. – Crit Rev Oral. Biol. 2002;13:238 75. 35. Gonzalez OA, Novak MJ, Kirakodu S, Orraca L, Chen KC, Stromberg A, 12. Shi GP, Webb AC, Foster KE, Knoll JH, Lemere CA, Munger JS, Chapman HA. Gonzalez-Martinez J, Ebersole JL. Comparative analysis of gingival tissue Human cathepsin S chromosomal localization, gene structure, and tissue antigen presentation pathways in ageing and periodontitis. J Clin – distribution. J Biol Chem. 1994;269:11530 6. Periodontol. 2014;41:327–39. 13. Liuzzo JP, Petanceska SS, Moscatelli D, Devi LA. Inflammatory mediators 36. Wu H, Cheng XW, Hu L, Hao CN, Hayashi M, Takeshita K, Hamrah MS, Shi regulate cathepsin S in macrophages and microglia: A role in attenuating GP, Kuzuya M, Murohara T. Renin inhibition reduces atherosclerotic plaque – heparan sulfate interactions. Mol Med. 1999;5:320 33. neovessel formation and regresses advanced atherosclerotic plaques. 14. Petanceska S, Canoll P, Devi LA. Expression of rat cathepsin S in phagocytic Atherosclerosis. 2014;237:739–47. – cells. J Biol Chem. 1996;271:4403 9. 37. Garg G, Pradeep AR, Thorat MK. Effect of nonsurgical periodontal 15. Andrault PM, Samsonov SA, Weber G, Coquet L, Nazmi K, Bolscher JG, therapy on crevicular fluid levels of Cathepsin K in periodontitis. Arch Lalmanach AC, Jouenne T, Brömme D, Pisabarro MT, Lalmanach G, Lecaille Oral Biol. 2009;54:1046–51. F. Antimicrobial Peptide LL-37 Is Both a Substrate of Cathepsins S and K 38. Gruber R. Molecular and cellular basis of bone resorption. Wien Med – and a Selective Inhibitor of Cathepsin L. Biochemistry. 2015;54:2785 98. Wochenschr. 2015;165:48–53. – 16. Nakanishi H. Microglial functions and proteases. Mol Neurobiol. 2003;27:163 76. 39. Hao L, Chen J, Zhu Z, Reddy MS, Mountz JD, Chen W, Li YP. Odanacatib, A 17. Shi HT, Wang Y, Jia LX, Qin YW, Liu Y, Li HH, Qi YF, Du J. Cathepsin S Cathepsin K-Specific Inhibitor, Inhibits Inflammation and Bone Loss Caused contributes to macrophage migration via degradation of elastic fibre by Periodontal Diseases. J Periodontol. 2015;86:972–83. integrity to facilitate vein graft neointimal hyperplasia. Cardiovasc Res. 40. Barry ZT, Platt MO. Cathepsin S cannibalism of cathepsin K as a mechanism – 2014;101:454 63. to reduce type I collagen degradation. J Biol Chem. 2012;287:27723–30. 18. Wu H, Cheng XW, Hu L, Takeshita K, Hu C, Du Q, Li X, Zhu E, Huang Z, 41. Li X, Cheng XW, Hu L, Wu H, Guo-Ping HCN, Jiang H, Zhu E, Huang Z, Inoue Yisireyili M, Zhao G, Piao L, Inoue A, Jiang H, Lei Y, Zhang X, Liu S, Dai Q, A, Sasaki T, Du Q, Takeshita K, Okumura K, Murohara T, Kuzuya M. Cathepsin Kuzuya M, Shi GP, Murohara T. Cathepsin S Activity Controls Injury-Related S activity controls ischemia-induced neovascularization in mice. Int J Cardiol. Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K-Akt/p- 2015;183:198–208. – HDAC6 Signaling Pathway. Artioscler Thromb Vasc Biol. 2016;36:1549 57. 42. ChongCH,CarnesDL,MoritzAJ,OatesT,RyuOH,SimmerJ,CochranDL.Human 19. Zavasnik-Bergant V, Sekirnik A, Golouh R, Turk V, Kos J. periodontal fibroblast response to enamel matrix derivative, amelogenin, and Immunochemical localisation of cathepsin S, cathepsin L and MHC class platelet-derived growth factor-BB. J Periodontol. 2006;77:1242–52. II-associated p41 isoform of invariant chain in human lymph node 43. Hoang AM, Oates TW, Cochran DL. In vitro wound healing responses to – tissue. Biol Chem. 2001;382:799 804. enamel matrix derivative. J Periodontol. 2000;71:1270–7. 20. Memmert S, Damanaki A, Nogueira AVB, Eick S, Nokhbehsaim M, 44. Aukhil I. Biology of wound healing. Periodontol. 2000;22:44–50. Papadopoulou AK, Till A, Rath B, Jepsen S, Götz W, Piperi C, Basdra EK, Cirelli